All mammals except humans and certain other primates contain glycoproteins that have terminal alpha-1,3-galactosyl (alpha-gal) glycan structures resulting from the activity of the enzyme alpha-1,3-galactosyl transferase (Galili et al, J Biol Chem, 263:33, 1988). The enzyme adds a galactose residue to terminally located beta-1,4-linked galactose residues. Beta-1,4-linked galactose residues are found at the termini of many N-glycans of mammals, including those of humans.
The human immune system has adapted natural immunity to quickly respond to the presence of terminal alpha-gal residues. Approximately one percent of circulating IgGs are directed against the alpha-1,3-galactose epitope (Galili et al, Blood, 82:8, 1993). Antigens that exhibit this epitope are recognized by circulating antibodies, resulting in complement activation, and the efficient activation of antigen-presenting cells via an Fcγ receptor-mediated pathway and the stimulation of a cytotoxic T-cell response. Targeting an immune complex to antigen presenting cells has been shown to reduce the amount of antigen required to elicit a T-cell response.
Current recombinant vaccines generally suffer from a lack of specific immunogenic response. Furthermore, current vaccines often require general immune stimulators known as adjuvants to elicit a sustained cytotoxic T-cell response. In a limited demonstration, a protein-based vaccine with N-glycans containing terminal α-1,3-galactose has been reported to improve the immunogenicity of such a molecule. (Abdel-Motal et al, J Virology, 80:14, 2006; Abdel-Motal et al, J Virology, 81:17, 2007.) This may be because humans have a high level of circulating antibodies directed against α-1,3-galactose residues. Moreover, because these proteins may be able to stimulate antigen presentation via antibody-directed Fc-gamma mediated signaling, they may reduce the need for non-specific adjuvants. The current state of the art only allows for production of such a vaccine by producing the protein with terminal sialic acid structures (e.g. NANA), then removing the NANA residues in vitro by enzymatic digest to expose the β-1,4 linked galactose residues, and subsequently adding terminal α-1,3-galactose through enzymatic addition (see, Galili, U.S. Pat. No. 6,361,775). This process is expensive, cumbersome, and not easily scalable. Attempts have been made to produce alpha-gal epitopes on viral proteins such as influenza virus hemagglutinin; Henion et al, Vaccine, 15:1174-1182 (1997); gp120; Abdel-Motal et al., J. Virology, 80:6943-6951 (2006); and on cancer cells; Unfer et al., Cancer Res., 63:987-993 (2003).